Nucleobase‐Caged Phosphoramidites for Oligonucleotide Synthesis

Alexander Heckel1

1 University of Bonn, Bonn
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.17
DOI:  10.1002/0471142700.nc0117s29
Online Posting Date:  June, 2007
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Abstract

Caged compounds are biologically active agents bearing a photolabile group in a strategic position, which makes them temporarily inactive. These compounds can then be delivered to a biological sample without immediately generating an effect. When the sample is then irradiated, e.g., with a laser in a (confocal) microscope, the activity of the substance is released with exact spatiotemporal and dose control. This unit deals with the synthesis of protected nucleoside phosphoramidites bearing a caging group on the nucleobase, which prevents the nucleobases from forming normal Watson‐Crick base pairs. These amidites can be used to generate caged oligodeoxynucleotides with a transitory local perturbation that adds an element of spatiotemporal control to oligonucleotide‐based applications.

Keywords: caging; temporary mismatch; spatiotemporal control; photoactivation; light‐trigger

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Protected NPE‐ and NPP‐Caged Thymidine Phosphoramidites
  • Basic Protocol 2: Preparation of a Protected NPP‐Caged Deoxyguanosine Phosphoramidite
  • Basic Protocol 3: Preparation of a Protected NPE‐Caged Deoxycytidine Phosphoramidite
  • Support Protocol 1: Preparation of the Caging Precursor 1‐(2‐Nitrophenyl)ethan‐1‐ol
  • Support Protocol 2: Preparation of the Caging Precursor 2‐(2‐Nitrophenyl)propan‐1‐ol
  • Support Protocol 3: Preparation of the Caging Precursor 1‐(2‐Nitrophenyl)ethylamine
  • Support Protocol 4: Preparation of (4‐Isopropylphenoxy)acetyl Chloride for Protection of Deoxyguanosine
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Protected NPE‐ and NPP‐Caged Thymidine Phosphoramidites

  Materials
  • Thymidine (S.4)
  • Acetic anhydride (Ac 2O)
  • Pyridine
  • Hexane
  • Acetone
  • Chloroform
  • 1 M aqueous sulfuric acid, 4°C
  • Saturated aqueous NaHCO 3
  • Anhydrous Na 2SO 4
  • Diethyl ether
  • Ethanol
  • Acetonitrile
  • Argon source
  • 4‐(N,N‐Dimethylamino)pyridine (DMAP)
  • Triethylamine (Et 3N)
  • 2,4,6‐Triisopropylbenzenesulfonyl chloride (iPr 3C 6H 2SO 2Cl)
  • Cyclohexane
  • 1‐(2‐Nitrophenyl)ethan‐1‐ol (NPE‐OH; S.22; see protocol 4) or 2‐(2‐nitrophenyl)propan‐1‐ol (NPP‐OH; S.24; see protocol 5)
  • 1 M aqueous HCl
  • Anhydrous MgSO 4
  • Methanol (MeOH)
  • 33% (v/v) aqueous ammonium hydroxide solution (NH 4OH)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Dichloromethane (CH 2Cl 2)
  • N,N‐Diisopropylethylamine (DIPEA, Hünig's base)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • 50‐, 100‐, and 250‐mL round‐bottom flasks
  • Reflux condenser
  • 90°C oil bath
  • Rotary evaporator connected to vacuum pump
  • High vacuum system
  • 500‐µL syringes
  • Separatory funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Preparation of a Protected NPP‐Caged Deoxyguanosine Phosphoramidite

  Materials
  • 2′‐Deoxyguanosine (S.9)
  • Pyridine
  • N,N‐Dimethylformamide (DMF)
  • Imidazole
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl)
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH)
  • Ethanol (EtOH), cold
  • (4‐Isopropylphenoxy)acetyl chloride (iPrPacCl; S.30; see protocol 7)
  • Cyclohexane
  • Acetone
  • Tetrahydrofuran (THF)
  • 2‐(2‐Nitrophenyl)propan‐1‐ol (NPP‐OH; S.24; see protocol 5)
  • Triphenylphosphine
  • 40% diethylazodicarboxylate (DEAD) in toluene
  • Ethyl acetate
  • Brine (saturated NaCl)
  • Anhydrous MgSO 4
  • Glacial acetic acid
  • 1 M tetrabutylammonium fluoride (TBAF) in THF
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Triethylamine (Et 3N)
  • N,N‐Diisopropylethylamine (DIPEA, Hünig's base)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Saturated aqueous NaHCO 3
  • Dry acetonitrile
  • 50‐, 100‐, and 250‐mL round‐bottom flasks
  • Rotary evaporator connected to a vacuum pump
  • High vacuum
  • Büchner funnel connected to a vacuum pump
  • Water aspirator
  • Syringes
  • Separatory funnels
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of a Protected NPE‐Caged Deoxycytidine Phosphoramidite

  Materials
  • 2′‐Deoxyuridine (S.15)
  • Pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH)
  • Ethanol (EtOH)
  • Ethyl acetate
  • Saturated aqueous NaHCO 3
  • Brine (saturated NaCl)
  • Anhydrous MgSO 4
  • n‐Hexane
  • 4‐(N,N‐Dimethylamino)pyridine (DMAP)
  • Acetic anhydride (Ac 2O)
  • Triethylamine (Et 3N)
  • 5% (w/v) aqueous citric acid
  • 2‐Mesitylenesulfonyl chloride (Me 3C 6H 2SO 2Cl)
  • N,N‐Dimethylformamide (DMF)
  • 1‐(2‐Nitrophenyl)ethylamine (NPE‐NH 2; S.26; see protocol 6)
  • 33% methylamine in ethanol
  • N,N‐Diisopropylethylamine (DIPEA, Hünig's base)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Cyclohexane
  • Acetone
  • Acetonitrile
  • 50‐mL, 250‐mL, and 1‐L round‐bottom flasks
  • Rotary evaporator connected to a vacuum pump
  • Glass sinter filter
  • High vacuum
  • Syringes
  • Reflux condenser
  • 90°C oil bath
  • Silicone septa, optional
  • Separatory funnel
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Preparation of the Caging Precursor 1‐(2‐Nitrophenyl)ethan‐1‐ol

  Materials
  • 2‐Nitroacetophenone (S.21)
  • 1,4‐Dioxane
  • Methanol (MeOH)
  • Sodium borohydride (NaBH 4)
  • Dichloromethane (CH 2Cl 2)
  • Anhydrous MgSO 4
  • 250‐mL round‐bottom flask
  • Rotary evaporator connected to a vacuum pump
  • Separatory funnel
  • Additional reagents and equipment for TLC ( appendix 3D)

Support Protocol 2: Preparation of the Caging Precursor 2‐(2‐Nitrophenyl)propan‐1‐ol

  Materials
  • 1‐Ethyl‐2‐nitrobenzene (S.23)
  • Dimethylsulfoxide (DMSO)
  • Paraformaldehyde
  • 40% Triton B (BuMe 3NOH) in methanol
  • Cyclohexane
  • Acetone
  • 100‐mL round‐bottom flask
  • Reflux condenser
  • 90°C oil bath
  • Distillation apparatus
  • Additional reagents and equipment for TLC ( appendix 3D)

Support Protocol 3: Preparation of the Caging Precursor 1‐(2‐Nitrophenyl)ethylamine

  Materials
  • 1‐Ethyl‐2‐nitrobenzene (S.23)
  • Tetrachlorocarbon (CCl 4)
  • N‐Bromosuccinimide (NBS)
  • Dibenzoyl peroxide [(BzO) 2]
  • Hexane
  • Acetone
  • Cyclohexane
  • N,N‐Dimethylformamide (DMF)
  • Potassium phthalimide
  • Ethyl acetate
  • Dichloromethane (CH 2Cl 2)
  • Saturated aqueous NaHCO 3
  • Anhydrous MgSO 4
  • Ethanol
  • 80% hydrazinium hydroxide solution in water
  • Methanol
  • Triethylamine (Et 3N)
  • 100‐ and 500‐mL round‐bottom flasks
  • Reflux condensers
  • 60°, 77°, and 155°C oil baths
  • Büchner funnel connected to a vacuum pump
  • Rotary evaporator connected to a vacuum pump
  • Separatory funnels
  • High vacuum
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 4: Preparation of (4‐Isopropylphenoxy)acetyl Chloride for Protection of Deoxyguanosine

  Materials
  • 60% NaH in paraffin
  • Tetrahydrofuran (THF)
  • 4‐Isopropylphenol (S.27)
  • Bromoacetic acid methyl ester
  • Diethyl ether
  • Anhydrous MgSO 4
  • 1,4‐Dioxane
  • Lithium hydroxide
  • Half‐concentrated HCl
  • Ethyl acetate
  • Thionyl chloride (SOCl 2)
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Dropping funnel
  • Rotary evaporator connected to a vacuum pump
  • Separatory funnels
  • Reflux condenser
  • 90°C oil bath
  • Distillation bridge
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Figures

Videos

Literature Cited

Literature Cited
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